RESUMO
A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera's database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity > or =90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000-20 000 NotI sites, of which 6000-9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.
Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Análise de Sequência de DNA/métodos , Linhagem Celular Transformada , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 22/genética , Ilhas de CpG/genética , DNA/química , DNA/genética , Bases de Dados de Ácidos Nucleicos , Genes/genética , Genoma Humano , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
We have identified a novel human gene UNC93B1 encoding a protein related to unc-93 of Caenorhabditis elegans. The combined sequence derived from several cDNA clones is 2282 bp and comparison with genomic sequence shows that the gene contains 11 exons. The longest open reading frame encodes a deduced sequence of 597 amino acids. Homology analysis shows that the hUNC93B1 gene is highly conserved and related to sequences in Arabidopsis thaliana, C. elegans, Drosophila melanogaster, chicken and mouse. Structural analysis of the deduced amino acid sequence of hUNC93B1 points to possible existence of multiple membrane-spanning domains. hUNC93B1 protein also displays some similarities to the bacterial ABC-2 type transporter signature and to ion transporters of Deinococcus radiodurans and Helicobacter pylori. As revealed by Northern analysis, the level of expression varies significantly between tissues, with the highest level detected in the heart. The gene was mapped to chromosomal band 11q13 by fluorescence in situ hybridization. We suggest that this gene is a member of a novel hUNC93B-related gene family.
Assuntos
Proteínas de Caenorhabditis elegans , Genes/genética , Proteínas de Membrana Transportadoras/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Feminino , Proteínas de Helminto/genética , Humanos , Hibridização in Situ Fluorescente , Proteínas de Membrana/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We have cloned a novel human mixed-lineage kinase gene, MLK4. Two alternatively spliced forms, MLK4α (580 aa) and MLK4ß (1036 aa), have been identified and mapped to chromosomal band 1q42. MLK4 shows high amino acid homology to the kinase catalytic domain of MLK3 (72%), MLK1 (71%) and MLK2 (69%). Strong expression of MLK4 was detected in the human pancreas and kidneys. pCMV-MLK4ß c-myc-tagged protein (human) was expressed in the cytoplasm and nucleus of transiently transfected COS-1 cells, while pCMV-MLK4α c-myc-tagged protein (human) was expressed in cytoplasm only. Both MLK4 isoforms reduced the colony formation ability of MCF7 cells by 85%-95% and almost totally suppressed cell proliferation in the CyQUANT cell proliferation assay. Human pCMV-MLK4ß transgenic mice expressed the MLK4ß in all tissues examined but no phenotypic abnormalities were observed. Thus, in this work, we present the cloning and sequencing of MLK4α and MLK4ß for the first time; the data obtained suggest that MLK4 may function as a MAP kinase.